Peripheral blood in the circulatory system of a human or animal is comprised principally of red blood cells, i.e. erythrocytes, and white blood cells, i.e. leukocytes. The variety of functions of leukocytes and their clinical relevance has generated great interest in the scientific community. The family of white blood cells is comprised of neutrophils, monocytes, eosinophils, basophils and lymphocytes. Lymphocytes are of T-lymphocyte and B-lymphocyte types which have numerous subsets. Neutrophils, eosinophils and basophils are known as "granulocytes" because of their content of cytoplasmic granules.
Neutrophils, monocytes, eosinophils and basophils are known as phagocytes because their primary function in the human immune system is to phagocytize or ingest bacteria, microorganisms and other types of foreign materials. These cells are produced from common progenitor cells in the bone marrow of a human or animal and are known to circulate in peripheral blood and finally, enter tissues as necessary for control of infection or to participate in any type of inflammatory reaction. However, each of these phagocytes has different functions and behaves as a related but separate system.
The neutrophil is the most common leukocyte in human and animal peripheral blood. One microliter of normal human whole blood includes, on average, 5.times.10.sup.3 leukocytes of which 3,075 are neutrophils, 150 are eosinophils, 25 are basophils, 250 are monocytes, and 1,500 are lymphocytes.
In the response of granulocytes or mononuclear phagocytes to any type of infection or inflammation, these cells are activated first to migrate to the appropriate area in response to chemoattractant factors, such as, certain bacterial products, complement component, and other factors. This attraction process is termed "chemotaxis". Once in an area of inflammation or infection, granulocytes and mononuclear phagocytes must establish a firm attachment to their targets. For this purpose, these cells possess a number of specific cell surface receptor glycoproteins that promote this interaction, such as complement, Fc, and fibronectin receptors.
A very important family of cell surface receptor glycoproteins is the leukocyte cell adhesion molecular (LEU-CAM) family (CD11/CD18). This family is comprised of at least three (3) cell surface proteins which have two (2) subunits each. They share a common beta subunit of 94,000 dalton molecular weight (CD18), and have different alpha subunits. The known members of this family are termed LFA-1 (CD 11a/CD18), Mol (CD 11b/CD18), and P150,94 (CD 11c/CD18) which evidence alpha subunits of 180,000, 155,000 and 150,000 dalton molecular weight, respectively. Each of these cell surface proteins has been specifically identified through the use of monoclonal antibodies. The biological importance of this family of surface glycoproteins has been recognized through the identification of a human disease in which leukocytes are genetically deficient in this family of antigens. (Arnaout, M.A., Dana, N., Pitt, J., and Todd, R.F. III., Deficiency of two human leukocyte surface membrane glycoproteins (Mol and LFA-1). Fed. Proc. 44: 2664-2670 (1985). The disease is characterized by recurrent severe bacterial infections and deficiencies in adhesion-dependent functions such as phagocytosis, neutrophil spreading on plastic, leukoaggregation, and chemotaxis.
The Mol glycoprotein has been of particular interest as it has been shown that this particular structure has the capacity to bind a component of complement termed iC3b, a fragment of the third component of complement. Arnaout, M.A., Todd, R.F. III, Dana, N., Melamed, J., Schlossman, S.F., and Colten, H.R., Inhibition of phagocytosis of complement C3 or IgG-coated particles and of iC3b binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol), J. Clin, Invest. 72:171-179 (1983). Also, the Mol glycoprotein is critically important in all of the adhesion-dependent phagocyte functions. Different monoclonal antibodies have been shown to inhibit the functions of the Mol glycoprotein.
Mol is a cell surface glycoprotein present on granulocytes, mononuclear phagocytes and null cells. Todd, R.F.III, Nadler, L.M. and Schlossman, S.F., Antigens on Human Monocytes, Journal of Immunology, 126: 1435-1442 (1981). In humans, this molecule consists of two non-covalently linked proteins of 155,000 and 94,000 daltons. Todd, R.F. III, van Agthoven, A., Schlossman, S.F., and Terhorst, C. Structural analysis of differentiation antigens. Mol and Mo2 on human monocytes, Hybridoma 1:329-337 (1982). This complex has been shown to mediate cell adhesion to a variety of surfaces including other granulocytes, endothelium, and inert substrates. Genetic deficiencies in these molecules result in recurrent bacterial infections due to the inability of granulocytes to mediate an anti-microbial inflammatory response. Patients who are deficient in these molecules are characterized by an elevated leukocyte count (called "leukocytosis" and functional defects in phagocyte activity as measured in vitro by reduced or absent aggregation adhesion to substrates, chemotaxis, and phagocytosis of opsonized particles. Activation of granulocytes and monocytes by soluble inflammatory mediators increases expression of these molecules. Todd, R.F. III, Arnaout, M.A., Rosin, R.E., Crowley, C.A., Peters, W.A. and Babior, B.M. The subcellular localization of Mol (Mol.alpha.; formerly gp.sup.110) a surface glycoprotein associated with neutrophil adhesion. J. Clin. Invest. 74:1280-1290 (1984); Arnaout, M.A., Hakim, R.M., Todd, R.F., Dana, N. and Colten, H.R., Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis, New Engl. J. Med., 312: 457-462 (1985). Monoclonal antibodies directed against the Mol glycoprotein effectively prevent neutrophil aggregation in vitro as well as prevent phagocytosis. In a rat lung model of neutrophil-mediated lung injury (acute respiratory distress syndrome [ARDS]), anti-Mol monoclonal antibody significantly inhibited plumonary endothelial damage produced by activated human neutrophils. Ismail, G., Morganroth, H.L., Todd, R.F. III, and Boxer, L.A. Prevention of pulmonary injury in isolated perfused rat lungs by activated human neutrophils preincubated with anti-Mol monoclonal antibody, Blood 69:1167-1174, (1987).
While the inflammatory response of leukocytes is vital to the eradiction of invading microorganisms, a substantial and convincing body of evidence indicates that inflammatory phagocytes cause damage to various organs and tissues when these cells are activated in vivo by soluble inflammatory factors that are generated by inciting pathological events. Harlan, J. M., Leukocyte-Endothelial Interactions, Blood 65: 513-525 (1985). The adhesion and spreading of activated neutrophils and mononuclear phagocytes to vascular endothelial cells with the subsequent release of toxic oxidative metabolites and proteases has been implicated in the organ damage observed in diseases, such as, adult respiratory distress syndrome (ARDS; shock lung syndrome), glomerulonephritis, and inflammatory injury occurring after reperfusion of ischemic tissue such as to the heart, bowel, and central nervous system. Reviewed in Harlan, J. M., ibid.) That the heart muscle or myocardium is vulnerable to the inflammatory response of activated leukocytes has been demonstrated by the outcome of several investigations. These studies have demonstrated that if dogs are depleted of circulating granulocytes with a neutrophil specific antiserum [Romson, J. L., et al., Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog, Circulation, 67: 1016-1023 (1983)] or nitrogen mustard [Mullane, K. M. et al., Role of leukocytes in acute myocardial infarction in anesthetized dogs: Relationship to myocardial salvage by anti-inflamatory drugs, J. Pharmacal. Exp. Ther. 228: 510-522 (1984)] prior to the induction of regional myocardial ischemia and reperfusion, the size of myocardial infarct that results is significantly smaller compared to dogs with normal circulating neutrophil counts. There are a number of other studies that have shown that agents that inhibit neutrohpil activation also result in reduced myocardial infarct size. Romson, J. L. et al., The effect of ibuprofen on accumulation of 111-indium labelled platelets and leukocytes in experimental myocardial infarction, Circulation 66: 1002-1011 (1982); Bednar, M. et al., Nafazatrom-induced salvage of ischemic myocardium in anesthetized dogs is mediated through inhibition of neutrophil function, Circ. Res. 57 : 131-141 (1985).
One monoclonal antibody which evidences the capability of inhibiting adhesion-dependent functions but does not affect binding of iC3b is known as MY904. Dana, N., Styrt, B., Griffin, J. D., Todd, R. F. III, Klempner, M. S., and Arnaout, M. A., Two functional domains in the phagocyte membrane glycoprotein Mol identified with monoclonal antibodies, J. Immunol. 137:3259-3263 (1986). Thus, the binding of the monoclonal antibody MY904 to neutrophils could specifically inhibit migration of neutrophils to an area of inflammation or infection. Further, such specific binding of MY904 antibody could inhibit the adhesion and spreading of activated neutrophils reaching such an area and then block the deleterious effects of toxic substances released by the granulocyte.
The method embodying the invention utilizes the specific advantages of the MY904 monoclonal antibody for reducing injury in vivo. The MY904 monoclonal antibody is administered in vivo in the setting of an acute inflammatory response mediated by inflammatory leukocytes, for example, in an acute coronary thrombosis experience just prior to the restoration of myocardial blood flow to ischemic myocardium. This infusion of MY904 antibody to impact on the phagocyte population in peripheral blood or tissue may inhibit or diminish the ability of these inflammatory cells to mirgrate to the inflammatory site within the affected tissue; and further, may inhibit adhesion of neutrophils, for instance, in such area so as to inhibit or minimize the potential deleterious effects of toxic substances released by adherent cells. For example, this procedure was determined to materially reduce tissue damage in the area of myocardial infarction after myocardial blood flow is returned.